THE RAS-RELATED PROTEIN RAD ASSOCIATES WITH THE CYTOSKELETON IN A NON-LIPID-DEPENDENT MANNER

Rad is the prototypic member of a new family of Ras-related proteins ( Rad, Gem, and Kir) which lack typical C-terminal amino acid motifs for isoprenylation, In mouse C2C12 muscle cell Lines about 50% of Rad pro tein resides in the cytosol and behaves as a hydrophilic protein parti tioning away from TX-114. The remainder of Rad is associated with plas ma and internal membranes. The association of Rad with the membrane do es not occur through the lipid bilayer, but instead depends on the int eraction of Rad with the cytoskeleton or membrane skeleton. In contras t to Ras, biosynthetic labeling of cellular proteins in C2C12 cells wi th [H-3]palmitic acid demonstrates that Rad is not modified with this fatty acid, and inhibition of isoprenylation with lovastatin treatment has no effect on Rad subcellular distribution. Furthermore, removal o f the C-terminal 11 amino acids that are precisely conserved in all th ree Rad family members has no effect on Rad subcellular distribution. Addition of the 9 amino acids from the C-terminus of H-Ras to the trun cated Rad protein results in a redistribution of Rad from the cytosol to the membrane skeleton without the presence of any detectable lipid modification of the chimeric protein. These data suggest that Rad poss esses unique cellular localization signals which, in contrast to other Ras-related family members, do not depend on the lipid modification o f the C-terminus. (C) 1998 Academic Press.